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. 1996 Aug;118(8):2213–2219. doi: 10.1111/j.1476-5381.1996.tb15665.x

Characterization of the P2Y-purinoceptor involved in the ATP-induced rise in cytosolic Ca2+ concentration in rat ileal myocytes.

P Pacaud 1, E Feolde 1, C Frelin 1, G Loirand 1
PMCID: PMC1909881  PMID: 8864564

Abstract

1. The P2-purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2. The P2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1-receptor. 3. Short applications of the purinoceptor agonists induced a transient rise in [Ca2+]i in an all-or-nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2-methylthioATP = 2-chloro-ATP > ADP > ATP, with concentrations giving [Ca2+]i response in 50% of cells ranging between 3 nM and 0.6 microM. The concentration-response curves to ADP and ATP were shifted to the right by 10 microM pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 4. Although the rise in [Ca2+]i induced by stimulation of the ileal P2v-purinoceptor was inhibited by heparin (5 mg ml-1), we were not able to detect stimulation of phospholipase C under conditions (37 degrees C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)-induced increase in InsP accumulation was suppressed when the agonist was applied at 20 degrees C while a CCh-induced [Ca2+]i rise similar to that obtained in response to the P2-purinoceptor agonist was still observed. 5. Our results indicate that the rat ileal myocytes express a PPADS-sensitive P2-purinoceptor similar to the P2Y1-receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+]i by activation of the inositol 1,4,5-trisphosphate receptor-channel of the intracellular Ca2+ store, indicating that they couple to phospholipase C.

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Selected References

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  1. Abbracchio M. P., Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther. 1994;64(3):445–475. doi: 10.1016/0163-7258(94)00048-4. [DOI] [PubMed] [Google Scholar]
  2. Blottière H. M., Loirand G., Pacaud P. Rise in cytosolic Ca2+ concentration induced by P2-purinoceptor activation in isolated myocytes from the rat gastrointestinal tract. Br J Pharmacol. 1996 Mar;117(5):775–780. doi: 10.1111/j.1476-5381.1996.tb15259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bo X., Zhang Y., Nassar M., Burnstock G., Schoepfer R. A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett. 1995 Nov 13;375(1-2):129–133. doi: 10.1016/0014-5793(95)01203-q. [DOI] [PubMed] [Google Scholar]
  4. Boyer J. L., Downes C. P., Harden T. K. Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides. J Biol Chem. 1989 Jan 15;264(2):884–890. [PubMed] [Google Scholar]
  5. Boyer J. L., Lazarowski E. R., Chen X. H., Harden T. K. Identification of a P2Y-purinergic receptor that inhibits adenylyl cyclase. J Pharmacol Exp Ther. 1993 Dec;267(3):1140–1146. [PubMed] [Google Scholar]
  6. Boyer J. L., Zohn I. E., Jacobson K. A., Harden T. K. Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors. Br J Pharmacol. 1994 Oct;113(2):614–620. doi: 10.1111/j.1476-5381.1994.tb17034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brake A. J., Wagenbach M. J., Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature. 1994 Oct 6;371(6497):519–523. doi: 10.1038/371519a0. [DOI] [PubMed] [Google Scholar]
  8. Brown C., Tanna B., Boarder M. R. PPADS: an antagonist at endothelial P2Y-purinoceptors but not P2U-purinoceptors. Br J Pharmacol. 1995 Nov;116(5):2413–2416. doi: 10.1111/j.1476-5381.1995.tb15088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen C. C., Akopian A. N., Sivilotti L., Colquhoun D., Burnstock G., Wood J. N. A P2X purinoceptor expressed by a subset of sensory neurons. Nature. 1995 Oct 5;377(6548):428–431. doi: 10.1038/377428a0. [DOI] [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Filtz T. M., Li Q., Boyer J. L., Nicholas R. A., Harden T. K. Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. Mol Pharmacol. 1994 Jul;46(1):8–14. [PubMed] [Google Scholar]
  12. Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W., Harden T. K., Jacobson K. A., Leff P., Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev. 1994 Jun;46(2):143–156. [PMC free article] [PubMed] [Google Scholar]
  13. Frelin C., Breittmayer J. P., Vigne P. ADP induces inositol phosphate-independent intracellular Ca2+ mobilization in brain capillary endothelial cells. J Biol Chem. 1993 Apr 25;268(12):8787–8792. [PubMed] [Google Scholar]
  14. Furukawa K., Nomoto T. Postnatal changes in response to adenosine and adenine nucleotides in rat duodenum. Br J Pharmacol. 1989 Aug;97(4):1111–1118. doi: 10.1111/j.1476-5381.1989.tb12568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  16. Iino M., Yamazawa T., Miyashita Y., Endo M., Kasai H. Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells. EMBO J. 1993 Dec 15;12(13):5287–5291. doi: 10.1002/j.1460-2075.1993.tb06224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnson C. R., Hourani S. M. Contractile effects of uridine 5'-triphosphate in the rat duodenum. Br J Pharmacol. 1994 Dec;113(4):1191–1196. doi: 10.1111/j.1476-5381.1994.tb17123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Komori S., Bolton T. B. Calcium release induced by inositol 1,4,5-trisphosphate in single rabbit intestinal smooth muscle cells. J Physiol. 1991 Feb;433:495–517. doi: 10.1113/jphysiol.1991.sp018440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Komori S., Kawai M., Pacaud P., Ohashi H., Bolton T. B. Oscillations of receptor-operated cationic current and internal calcium in single guinea-pig ileal smooth muscle cells. Pflugers Arch. 1993 Sep;424(5-6):431–438. doi: 10.1007/BF00374905. [DOI] [PubMed] [Google Scholar]
  20. Lambrecht G., Friebe T., Grimm U., Windscheif U., Bungardt E., Hildebrandt C., Bäumert H. G., Spatz-Kümbel G., Mutschler E. PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses. Eur J Pharmacol. 1992 Jul 7;217(2-3):217–219. doi: 10.1016/0014-2999(92)90877-7. [DOI] [PubMed] [Google Scholar]
  21. Lewis C., Neidhart S., Holy C., North R. A., Buell G., Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature. 1995 Oct 5;377(6548):432–435. doi: 10.1038/377432a0. [DOI] [PubMed] [Google Scholar]
  22. Lustig K. D., Shiau A. K., Brake A. J., Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5113–5117. doi: 10.1073/pnas.90.11.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nicholls J., Hourani S. M., Kitchen I. The ontogeny of purinoceptors in rat urinary bladder and duodenum. Br J Pharmacol. 1990 Aug;100(4):874–878. doi: 10.1111/j.1476-5381.1990.tb14107.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pacaud P., Bolton T. B. Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells. J Physiol. 1991 Sep;441:477–499. doi: 10.1113/jphysiol.1991.sp018763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pacaud P., Loirand G., Baron A., Mironneau C., Mironneau J. Ca2+ channel activation and membrane depolarization mediated by Cl- channels in response to noradrenaline in vascular myocytes. Br J Pharmacol. 1991 Dec;104(4):1000–1006. doi: 10.1111/j.1476-5381.1991.tb12540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pacaud P., Loirand G., Grégoire G., Mironneau C., Mironneau J. Noradrenaline-activated heparin-sensitive Ca2+ entry after depletion of intracellular Ca2+ store in portal vein smooth muscle cells. J Biol Chem. 1993 Feb 25;268(6):3866–3872. [PubMed] [Google Scholar]
  27. Pirotton S., Raspe E., Demolle D., Erneux C., Boeynaems J. M. Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells. J Biol Chem. 1987 Dec 25;262(36):17461–17466. [PubMed] [Google Scholar]
  28. Tokuyama Y., Hara M., Jones E. M., Fan Z., Bell G. I. Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun. 1995 Jun 6;211(1):211–218. doi: 10.1006/bbrc.1995.1798. [DOI] [PubMed] [Google Scholar]
  29. Valera S., Hussy N., Evans R. J., Adami N., North R. A., Surprenant A., Buell G. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature. 1994 Oct 6;371(6497):516–519. doi: 10.1038/371516a0. [DOI] [PubMed] [Google Scholar]
  30. Webb T. E., Simon J., Krishek B. J., Bateson A. N., Smart T. G., King B. F., Burnstock G., Barnard E. A. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett. 1993 Jun 14;324(2):219–225. doi: 10.1016/0014-5793(93)81397-i. [DOI] [PubMed] [Google Scholar]
  31. Windscheif U., Pfaff O., Ziganshin A. U., Hoyle C. H., Bäumert H. G., Mutschler E., Burnstock G., Lambrecht G. Inhibitory action of PPADS on relaxant responses to adenine nucleotides or electrical field stimulation in guinea-pig taenia coli and rat duodenum. Br J Pharmacol. 1995 Aug;115(8):1509–1517. doi: 10.1111/j.1476-5381.1995.tb16644.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ziganshin A. U., Hoyle C. H., Lambrecht G., Mutschler E., Bümert H. G., Burnstock G. Selective antagonism by PPADS at P2X-purinoceptors in rabbit isolated blood vessels. Br J Pharmacol. 1994 Mar;111(3):923–929. doi: 10.1111/j.1476-5381.1994.tb14827.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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